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Imaging Antifungal Drug Molecules in Action using Soft X-Ray Tomography Print

 

Humankind has benefitted from a long and productive relationship with yeast. For example, fermentation by yeast is an essential step in the production of bread, beer, wine, and even biofuels. However, not all yeast are beneficial. One strain of yeast, Candida albicans, grows unnoticed on most peoples’ skin and in the intestines. In response to certain environmental conditions, C. albicans can switch to a pathogenic phenotype that causes infection. Yeast infections are commonplace, and in otherwise healthy individuals are usually treatable with over-the-counter medications; however, individuals with weakened immune systems can have very serious systemic consequences from a yeast infection. Treating systemic yeast infections is becoming increasingly difficult due to the growing number of yeast strains that have developed resistance to existing antimicrobial drugs. Consequently, there is a pressing need to develop new types of drugs capable of circumventing yeast drug-resistance mechanisms. To this end Stanford, University of California, San Francisco and LBNL researchers have used soft x-ray tomography to image the 3-D structure of both benign and infectious C. albicans yeast. They then imaged this yeast when treated with peptoids, a class of molecules that mimic the peptides our immune system uses as the first line of defense against microbial attack. Unlike conventional antimicrobials, microbes have yet to develop resistance mechanisms against peptides or peptoids, making them appealing candidates for pharmaceutical development.

Cell CAT Scans:
3-D Cell Imaging

Routine medical x rays, such as chest x rays, can be used to identify a number of diseases. But this technique only produces a 2-D image of the inside of the patient’s body, where detailed internal structures may be superimposed. To disentangle this information requires the use of tomographic methods that produce 3-D images. During tomography the specimen is imaged in 2-D from multiple angles around a rotation axis. These images, called a projection series, can be collected in less than three minutes per sample. The 2-D images are then aligned and a 3-D volumetric reconstruction of the specimen is calculated. Organelles and other sub-cellular structur es of interest can be automatically segmented (visually isolated from the rest of the cell) based on their x-ray absorption characteristics.

Soft x-ray tomography is an enormously powerful technique, similar to a medical CAT (computed axial tomography) scan, with applications in a number of diverse fields from material science to medicine. The research described here uses soft x-ray tomographic methods to image whole, hydrated yeast cells exhibiting various phenotypes (physical characteristics) at high spatial resolution. XM-2 is the first instrument in the world that enables tomographic methods to image cells with different phenotypes, or the sub-cellular results of treating disease-causing cells with candidate drug molecules.

See Berkeley Lab’s related article: A speedy CAT scan for cells.

The basic concept behind tomographic reconstruction: 2-D projection images of two yeast cells mounted in a glass capillary tube were taken at angular increments around a rotation axis. For clarity, projection images are shown at 30° intervals. In practice images are collected every 1 or 2° over a total range of 180°. A 3-D reconstruction of the yeast and the capillary tube (shown middle, top of the figure) is calculated either by ‘Filtered Back Projection’, or by algebraic reconstruction techniques. Sub-cellular structures of interest can be segmented (identified and visually differentiated) based on their x-ray absorption characteristics. [Image taken from G. McDermott, et al., Trends in Cell Biology 19, 587 (2009)]

To begin developing peptoids as antifungal drugs, researchers must determine how peptoids would function as therapeutic molecules. To obtain this insight, the Barron group from Stanford teamed up with Berkeley Lab’s National Center for X-Ray Tomography to carry out a series of imaging experiments on XM-2, the biological soft x-ray microscope at ALS Beamline 2.1. The team imaged the sub-cellular changes that take place when C. albicans becomes infectious. Once the team established the physical characteristics of benign and pathogenic forms of C. albicans they imaged the cells treated with peptoids.

The new microscope yielded stunningly clear 3-D images of the three phenotypes displayed by C. albicans – yeast-like, germ-tube and hyphal cells. Peptoid treatment suppresses formation of the pathogenic hyphal phenotype, and results in striking changes in cell and organelle morphology. In particular, peptoid treatment was seen to cause changes to the structures of the nucleus and nucleolus, and a significant increase in the size and location of lipid-like bodies in the cell, including inside the nucleus.

Treatment with peptoids yielded either a benign yeast-like phenotype, or a germ-tube that closely resembles the yeast-like cell. Segmented pathogenic hyphal cells (whose parts are identified and visually demarcated based on their x-ray absorption characteristics) showed marked structural differences, as compared to the yeast-like and germ-tube phenotypes.

Soft X-ray tomographic reconstruction of phenotypically distinct C. albicans cells. Volume rendered views from each tomographic reconstruction are shown for yeast-like (top), germ-tube (middle) and hyphal (bottom) cells; selected organelles have been segmented and color-coded for identification. Blue, nucleus; orange, nucleolus; gray, mitochondria; yellow, vacuole; green, lipid bodies. Scale bars, 1 μm (for top and middle), 2 μm (for bottom). Watch a video of these cells in 3-D. From Uchida et al. Proc. Nat. Acad. Sci. (USA) 106 (46), 19375 (2009).

 

C. albicans cells after treatment with antifungal peptoids 1 (top) and 2 (bottom). Treatment-induced structural changes in the nucleolus, and the incorporation of a large lipid body into the nucleus, can be seen, along with all segmented organelles. Watch a video of these cells in 3-D. Scale bars, 1 μm (both). From Uchida et al. Proc. Nat. Acad. Sci. (USA) 106 (46), 19375 (2009).

This is the first time it has been possible to view the consequences of a cell switching phenotypes, and of drug treatment on the detailed structure of a cell. This use of soft x-ray tomography is now generating considerable interest in the pharmaceutical industry as a potential method for reducing the cost and time it takes to get new drugs onto the market. XM-2 was the first soft x-ray microscope in the world to be designed for biological cryo-tomographic imaging, and its success has initiated the construction of a number of similar microscopes at synchrotrons around the world. Soft x-ray tomography stands alone as a technique for imaging the sub-cellular organization of fully hydrated eukaryotic cells without the use of stains or other contrast enhancing agents, allowing cells to be imaged close to their native states.

 


 

Research conducted by M. Uchida, G. McDermott, C. Knoechel (University of California, San Francisco); M. Wetzler and A.E. Barron (Stanford University School of Medicine); M. A. Le Gros, (Berkeley Lab); M. Mylls, (University of California, San Francisco and University of Jyväskylä, Finland); and C.A. Larabell (University of California, San Francisco and Berkeley Lab).

Research funding: National Institutes of Heath and U.S. Department of Energy, Office of Biological and Environmental Research. Operation of the ALS and of SSRL is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: M. Uchida, G. McDermott, M. Wetzler, M. A. Le Gros, M. Mylls, C. Knoechel, A. E. Barron, & C. A. Larabell, “Soft x-ray tomography of phenotypic switching and the cellular response to antifungal peptoids in Candida albicans,” Proc. Nat. Acad. Sci. (USA) 106 (46), 19375 (2009).

 

ALSNews Vol. 306